742
HETEROCYCLES, Vol. 86, No. 1, 2012
Synthesis of compound 9 (Click reaction of N-Boc-DL-propargylglycine (8) and benzyl azide (6))
Benzyl azide (6, 4.7 mg, 35.5 µmol) and N-Boc-DL-propargylglycine (8, 7.6 mg, 35.8 µmol) were
dissolved in tert-BuOH - water (1:1; 126 "L) including CuBr (0.52 mg, 3.6 µmol). The reaction mixture
was stirred at rt 4h, diluted with EtOAc and washed with brine. The aqueous solution was extracted with
EtOAc. The combined organic extract was dried over MgSO4, filtrated and concentrated. The residue was
subjected to silica gel chromatography (CH2Cl2 :MeOH = 4:1 then 2:1) to afford the product 9 as a pale
yellow amorphous mass (10.7 mg, 87%). 1H-NMR (CD3OD) !: 7.72 (1H, s), 7.39-7.29 (5H, m), 5.54 (2H,
13
s), 4.23 (1H, s), 3.24 (1H, dd, J = 14.5, 4.6 Hz), 3.07 (1H, dd, J = 14.5, 7.3 Hz), 1.37 (9H, s); C-NMR
(CD3OD) !: 157.57, 136.77, 129.96, 129.49, 129.06, 125.04 (broad), 80.32, 79.49, 54.88, 29.79, 28.68;
ESI-TOF-MS: [M+H]+ calculated for C17H23N4O4, 347.1719, found 347.1719.
Synthesis of compound 7 (Click reaction of DL-propargylglycine (3) and benzyl azide (6))
Benzyl azide (6, 4.7 mg, 35.5 µmol), DL-propargylglycine (3, 4.1 mg, 36.2 µmol), and sodium ascorbate
(1.4 mg, 7.7 µmol) were dissolved in tert-BuOH : water (1 : 1; 126 "L) including CuBr (0.52 mg, 3.6
µmol). The reaction mixture was stirred at 75 # for 4 h, filtrated and washed with water and ether,
successively. The yield was determined by 1H-NMR and 13C-NMR. 1H-NMR (D2O containing 0.5% DCl)
!: 7.81(1H, s), 7.15-7.04 (5H, m), 5.34 (2H, s), 4.13 (1H, t, J = 5.9 Hz), 3.14 (2H, d, J = 6.3 Hz);
13C-NMR (D2O containing 0.5% DCl) !: 170.82, 134.61, 129.76, 129.58, 128.90, 127.08 (broad), 55.29,
52.69, 25.75; ESI-TOF-MS: [M+H]+ calculated for C12H15N4O2, 247.1195, found 247.1189.
Synthesis of compound 1 (Click reaction of DL-propargylglycine (3) and biotin-PEG-azide (2))
Biotin-PEG-azide (2, 15.0 mg, 26 µmol) and DL-propargylglycine (3, 5.9 mg, 52 µmol) and sodium
ascorbate (1.0 mg, 5.2 µmol) were suspended in DMF : H2O (1 : 7; 300 "L) including of CuBr (0.4 mg,
2.6 µmol). The reaction mixture was stirred for 5 h at 75 # and concentrated. The residue was subjected
to silica gel chromatography (H2O :MeOH = 9:1) to afford product as pale yellow amorphous mass (16.4
mg, 90%). 1H-NMR (D2O) !: 7.79 (1H, s), 4.59 (1H, m), 4.47 (2H, m), 4.28 (1H, m), 3.91 (1H, t, J = 4.9
Hz), 3.83 (2H, t, J = 4.9 Hz), 3.57-3.49 (22H, m), 3.48 (2H, s), 3.24 (2H, t, J = 5.3 Hz), 3.19 (1H, m),
2.88 (1H, dd, J = 12.9, 4.9 Hz), 2.63 (1H, d, J = 12.9 Hz), 2.13 (2H, t, J = 7.1 Hz), 1.60-1.24 (6H, m);
13C-NMR (D2O) !: 177.54, 176.63 (broad), 165.97, 125.65 (broad), 70.68-69.86 (12C), 69.57, 69.43,
62.76, 60.92, 56.05, 50.63, 40.40, 39.58, 36.14, 28.59, 28.39, 25.83; 1carboxy, 2triazole missing
ESI-TOF-MS: [M+H]+ calculated for C29H52N7O10S, 690.3496, found 690.3517.
Cu(I) analysis in the click reaction with bathocuproine disulphonic acid
A fraction of the click reaction (9 µL) was pipetted out and added 17 mM bathocuproine disulphonic acid
aqueous solution (15 µL) and sodium acetate solution (1.22 M, 840 µL). The assay mixture was adjusted
to 1 mL with distilled water, then subjected to absorption measurement at 485 nm with
spectrophotometer.